Mediate Leukocyte Recruitment Research Articles

The role of junctional adhesion molecule‐C (JAM‐C) in oxidized LDL‐mediated leukocyte recruitment

The junctional adhesion molecule-C (JAM-C) was recently shown to be a counter receptor for the leukocyte beta2-integrin Mac-1 (CD11b/CD18), thereby mediating interactions between vascular cells, particularly in inflammatory cell recruitment. Here, we investigated the role of JAM-C in oxidized low-density lipoprotein (LDL)-mediated leukocyte recruitment. As compared with normal arteries, immunostaining of atherosclerotic vessels revealed a high expression of JAM-C in association with neointimal smooth muscle cells and the endothelium. Moreover, JAM-C was strongly up-regulated in the spontaneous early lesions in ApoE -/- mice. In vitro, cultured human arterial smooth muscle cells (HASMC) were found to express JAM-C, and oxLDL, as well as enzymatically modified LDL (eLDL) significantly up-regulated JAM-C on both HASMC and endothelial cells in a time- and dose-dependent manner. Although under quiescent conditions, JAM-C predominantly localized to interendothelial cell-cell contacts in close proximity to zonula occludens-1 (ZO-1), oxLDL treatment induced a disorganization of JAM-C localization that was no more restricted to the interendothelial junctions. JAM-C thereby mediated both leukocyte adhesion and leukocyte transendothelial migration upon oxLDL treatment of endothelial cells, whereas JAM-C on quiescent endothelial cells only mediates leukocyte transmigration. Thus, upon oxLDL stimulation endothelial JAM-C functions as both an adhesion, as well as a transmigration receptor for leukocytes. Taken together, JAM-C is up-regulated by oxLDL and may thereby contribute to increased inflammatory cell recruitment during atherosclerosis. JAM-C may therefore provide a novel molecular target for antagonizing interactions between vascular cells in atherosclerosis.

Analysis of NK cells and chemokine receptors in tumor infiltrating CD4 T lymphocytes in human renal carcinomas.

Recent data suggest that chemokines and chemokine receptors mediate leukocyte recruitment of all components of the antitumor response. This study aimed to phenotypically characterize the immune lymphocyte infiltrate in human renal cell carcinomas RCCs and at the invasive margin (tumor-host interface) and to define the association of these findings with established prognostic indicators. Tumor infiltrating lymphocytes TILs were obtained from 24 patients with RCC undergoing radical nephrectomy. Peripheral blood cells from 37 patients were also obtained before surgery. Our findings are consistent with the preferential recruitment of CD4+ Th1-polarized effector memory cells that express CXCR3/CCR5. These cells were the main component of TILs and expressed as CXCR3, CCR5, CD45RO, and CD95. Natural killer (NK) cells were found in significantly higher proportions in TILs of RCCs than in peripheral blood lymphocytes (PBLs) or in other tumors studied (colorectal and breast cancers), where these cells were found in small proportions. No differences in nuclear grade or other studied parameters were observed between the TILs and the lymphocytes present at the invasive margin, which showed a similar composition. However, differences were found according to the tumor stage. First, significantly fewer NK cells were observed in PBLs from metastatic patients. Second, a significantly lower proportion of CCR5/CXCR3/CD4+ cells and a higher proportion of CCR4/CD4+ cells were observed in metastatic patients, suggesting that preferential Th1-polarization may gradually change during the progression of renal cancer cells. Finally, the frequency of CD25/CD4+ cells was higher in metastatic patients. Although the sample of patients with metastasis was small, the overall results suggest a change in composition of the TILs that may potentially confer a selective advantage for tumor growth and may account for the suppression of an effective cytotoxic response.

Evaluation of cardioprotective effects of recombinant soluble P-selectin glycoprotein ligand-immunoglobulin in myocardial ischemia-reperfusion injury by real-time myocardial contrast echocardiography

The purpose was to examine the cardioprotective effects of recombinant P-selectin glycoprotein ligand-immunoglobulin (rPSGL-Ig) in ischemia-reperfusion injury by real-time myocardial contrast echocardiography (MCE). P-selectin mediates leukocyte recruitment into areas of inflammation. Sixteen pigs underwent 45 min of left anterior descending coronary artery occlusion followed by reperfusion and received rPSGL-Ig or vehicle. To assess changes in myocardial perfusion (A x beta), MCE was performed. After 120 min of reperfusion, A x beta in the risk area was higher (0.84 +/- 0.15 dB/s vs. 0.28 +/- 0.1 dB/s, p < 0.0001), and the infarct size was lower (30.3 +/- 12% vs. 57 +/- 14%, p = 0.002) in the rPSGL-Ig group compared with the vehicle group. Recombinant PSGL-Ig improved postischemic reflow accurately detected by real-time MCE.

Design and Synthesis of Leukocyte Adhesion Inhibitors

The recognition of glycoprotein ligands by the selectins mediates leukocyte recruitment during an inflammatory response. Mimetics whose designs are based on the sialyl Lewis-x tetrasaccharide, common to these glycoproteins, have proven to be good inhibitors of leukocyte binding to the selectins. Some of the more recent approaches to the synthesis of these mimetics, including the use of polymeric and liposomal displays, multicomponent condensation reactions, and solid-phase peptide synthesis, are presented.

α4β1 Integrin (VLA-4) Blockade Attenuates both Early and Late Leukocyte Recruitment and Neointimal Growth following Carotid Injury in Apolipoprotein E (–/–) Mice

Background: The α₄β₁ integrin (VLA-4) supports rolling and firm adhesion of leukocytes to inflamed tissues via ligation of VCAM-1 or fibronectin expressed on the activated endothelium. We tested the hypothesis that VLA-4 mediates leukocyte recruitment and neointimal growth after arterial injury in the atherosclerosis-prone apolipoprotein E (ApoE)-deficient mouse. Methods: ApoE (–/–) mice fed a Western diet underwent air desiccation injury, and the expression patterns of VLA-4 and VCAM-1 were determined by immunohistochemistry (IHC). To determine the effect of targeted VLA-4 blockade on leukocyte recruitment and neointimal growth, ApoE (–/–) mice received an intraperitoneal injection of a VLA-4 neutralizing monoclonal antibody (PS/2) at the time of injury alone or over a prolonged administration course. Additional mice received an isotype control antibody. Results: IHC demonstrated a marked increase in VLA-4 expression 7 days following injury. Prolonged administration of PS/2 resulted in a 72% reduction (p < 0.02) in neointimal growth 28 days following injury. IHC revealed a marked 95% reduction in neutrophil recruitment at 7 days and a 48% reduction in macrophage recruitment 28 days following injury with prolonged PS/2 administration. Conclusions: Prolonged VLA-4 blockade reduces leukocyte recruitment and neointimal growth following air desiccation injury in ApoE (–/–) mice. These findings demonstrate an important role for VLA-4 in the response to arterial injury.

Common chemokine receptor single nucleotide polymorphisms are associated with transplant vasculopathy in heart transplant recipients

Chemokines such as CX3CL1 (fractalkine), CCL2 (monocyte chemoattractant protein-1, MCP-1) and CCL5 (RANTES) have been extensively shown to be upregulated in transplanted hearts and to mediate leukocyte recruitment in transplant rejection. Chemokines are specifically recognized by leukocyte receptors allowing recruitment and migration of these cells to the site of inflammation. Recently single nucleotide polymorphisms (SNPs) in some chemokine receptors have been shown to alter leukocyte recruitment and have been linked to atherosclerosis. We therefore analyzed their importance in a cardiac transplant recipient (CTR) cohort and their association with transplant vasculopathy (TV).

Overlapping Roles of Endothelial Selectins and Vascular Cell Adhesion Molecule-1 in Immune Complex-Induced Leukocyte Recruitment in the Cremasteric Microvasculature

Many adhesion molecule pathways have been invoked as mediating leukocyte recruitment during immune complex-induced inflammation. However the individual roles of these molecules have not been identified via direct visualization of an affected microvasculature. Therefore, to identify the specific adhesion molecules responsible for leukocyte rolling and adhesion in immune complex-dependent inflammation we used intravital microscopy to examine postcapillary venules in the mouse cremaster muscle. Wild-type mice underwent an intrascrotal reverse-passive Arthus model of immune complex-dependent inflammation and subsequently, leukocyte-endothelial cell interactions and P- and E-selectin expression were assessed in cremasteric postcapillary venules. At 4 hours, the reverse-passive Arthus response induced a significant reduction in leukocyte rolling velocity and significant increases in adhesion and emigration. P-selectin expression was increased above constitutive levels whereas E-selectin showed a transient induction of expression peaking between 2.5 to 4 hours and declining thereafter. While E-selectin was expressed, rolling could only be eliminated by combined blockade of P- and E-selectin. However, by 8 hours, all rolling was P-selectin-dependent. In contrast, inhibition of vascular cell adhesion molecule-1 had a minimal effect on leukocyte rolling, but significantly reduced both adhesion and emigration. These observations demonstrate that immune complex-mediated leukocyte recruitment in the cremaster muscle involves overlapping roles for the endothelial selectins and vascular cell adhesion molecule-1.

Relative roles of ICAM-1 and VCAM-1 in the pathogenesis of experimental radiation-induced intestinal inflammation

Purpose Cell adhesion molecules mediate leukocyte recruitment into the irradiated organs; modulation of this process may protect from radiation damage. Our objective was to characterize the requirement for intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in intestinal inflammatory response after abdominal irradiation. Methods and materials Endothelial ICAM-1 and VCAM-1 expression was determined using radiolabeled antibodies in mice 24 h and 14 days after irradiation with 10 Gy, or sham radiation. Leukocyte–endothelial cell interactions in intestinal venules were assessed using intravital microscopy, and the function of ICAM-1 and VCAM-1 in this process by using blocking antibodies and ICAM-1 −/− mice. Results The number of adherent leukocytes significantly increased 24 h after irradiation and remained elevated at 14 days. Treatment with anti–ICAM-1 antibodies and ICAM-1 genetic deficiency significantly reduced leukocyte adhesion 24 h after irradiation. At 14 days after irradiation, both wild-type and ICAM-1 −/− mice had an upregulation of VCAM-1, expression, and VCAM-1 immunoneutralization, but not ICAM-1 immunoneutralization, significantly reduced leukocyte adhesion. In ICAM-1 −/− mice, regeneration of the intestinal epithelium was enhanced relative to wild-type mice. Conclusions ICAM-1 plays a key role in leukocyte recruitment at early time points after abdominal irradiation, whereas VCAM-1 is the main molecular determinant of leukocyte recruitment at late time points.

Chemokines and chemokine receptors are involved in the resolution or progression of renal disease

Locally secreted chemokines mediate leukocyte recruitment during the initiation and amplification phase of renal inflammation. In turn, the infiltrating leukocytes contribute to renal damage by releasing inflammatory and profibrotic factors. Rapid down modulation of the chemokine signal will support resolution of acute inflammation, whereas progression occurs if ongoing or repeated renal injury maintains continuous local chemokine secretion and leukocyte influx into the glomerulus or the interstitial space. In glomerular injury proteinuria itself as well as glomerular secreted cytokines stimulate downstream tubular epithelial cells to also secrete chemokines. During primary tubular injury, tubular epithelial cells directly become a major site of chemokine production. This in turn supports leukocyte infiltration and activation. Infiltrating leukocytes stimulate fibroblast proliferation and matrix synthesis, leading to widening of the interstitial space. The specific and intricate renal vascular architecture renders the organ susceptible to ischemic damage as interstitial volume increases. Ischemia in turn serves as a stimulus for chemokine and cytokine production and matrix synthesis. The mutual stimulation between fibroblasts and infiltrating leukocytes supports progressive tubular damage, renal fibrosis, and glomerulosclerosis. Potentially this vicious circle leading to progression of chronic nephropathies offers the opportunity for therapeutic intervention. Interfering with the chemokine network that mediates leukocyte recruitment may represent a promising therapeutic option for progressive renal disorders and renal fibrosis. This article summarizes the present data on the role of chemokines in acute and chronic renal disease with special emphasis on their potential role in mediating resolution or progression of renal disease as well as on therapeutic options.

Chemokine and chemokine receptor gene expression indicates acute rejection of human cardiac transplants.

Factors directing T-cell infiltration into allografts during acute rejection remain poorly defined. Chemokines have been shown to mediate leukocyte recruitment into allografts in animal models of rejection. The goal of this study was to test the presence and levels of chemokine and receptor gene expression in serial endomyocardial biopsy specimens from heart transplant patients and to correlate the levels observed with histopathologic rejection grade. Three hundred sixteen serial endomyocardial biopsy specimens from 30 heart transplant patients were obtained during the clinically scheduled surveillance heart biopsy program. The follow-up period was 1 year. The expression of interferon (IFN)-gamma inducible protein (IP)-10, monokine induced by IFN-gamma (Mig), interferon-inducible T-cell alpha chemoattractant (I-TAC), regulated on activation normal T-cell expressed and secreted (RANTES), monocyte chemotactic protein (MCP)-1, interleukin (IL)-8, and the receptors CXCR3 and CCR5 were tested using quantitative, real-time polymerase chain reaction. Biopsy samples were examined histologically to assign rejection grade. Expression of IP-10, Mig, I-TAC, RANTES, CXCR3, and CCR5, but not MCP-1 and IL-8, increased significantly in both grade 2 and grade 3 rejection (P</=0.0096). These increases returned to normal after treatment with pulse steroid therapy to treat the rejection episode. The expression of IP-10, Mig, I-TAC, RANTES, CXCR3, and CCR5 in cardiac allografts significantly correlates with the presence and grade of acute rejection.

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated fromTrypanosoma cruzitrypomastigotes induce in vivo leukocyte recruitment dependent on MCP-1 production by IFN-γ-primed-macrophages

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins from Trypanosoma cruzi trypomastigotes (tGPI-mucins) activate macrophages in vitro to produce proinflammatory cytokines, chemokines, and nitric oxide. These effects of tGPI-mucins may be important in the ensuing immune response to T. cruzi. Here, we have sought evidence for a role of tGPI-mucins in mediating leukocyte recruitment in vivo. tGPI-mucins are highly effective in promoting cell recruitment in the pleural cavity of mice primed with IFN-gamma-inducing agents but not in naïve mice. Maximal recruitment was observed at a dose between 250 and 1250 ng tGPI-mucins. There was a significant elevation in the levels of MCP-1 in the pleural cavity of primed animals injected with tGPI-mucins, and in vivo neutralization of MCP-1 abolished leukocyte recruitment. Pretreatment with anti-MIP-1alpha or anti-RANTES had no effect on the recruitment induced by tGPI-mucins. MCP-1 immunoreactivity was detected in pleural macrophages, and macrophages produced MCP-1 in vitro, especially after priming with IFN-gamma. Finally, tGPI-mucins induced significant leukocyte recruitment in primed C3H/HeJ but not in TLR2-deficient mice. Together, our results suggest that T. cruzi-derived GPI-mucins in conjunction with IFN-gamma may drive tissue chemokine production and inflammation and bear a significant role in the pathogenesis of Chagas disease.

Adhesion molecules in inflammatory diseases: insights from knockout mice.

Leukocyte/endothelial cell adhesion molecules are essential mediators of both immune and inflammatory responses. However, their specific roles in the initiation and progression of inflammatory diseases remain largely undefined. The focus of our laboratory is the identification of the adhesion molecule interactions that mediate leukocyte recruitment and tissue damage during the development of rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. For these studies, we use a basic genetic approach in mice, analyzing different gene-targeted adhesion molecule mutants, or "knockouts," in murine disease models. Our findings suggest that loss of intercellular adhesion molecule-1 significantly inhibits the development of arthritis and glomerulonephritis, while selectin deficiency results in accelerated development of joint and kidney inflammation. Our results also indicate that the beta2 integrins may play a key role in regulating the initiation of psoriasiform skin diseases.

Synthesis of C-X-C and C-C Chemokines by Human Peritoneal Fibroblasts: Induction by Macrophage-Derived Cytokines

Leukocyte accumulation during peritonitis is believed to be controlled by chemotactic factors released by resident peritoneal macrophages or mesothelial cells. Recent data indicate, however, that in many tissues fibroblasts play a key role in mediating leukocyte recruitment. We have therefore examined human peritoneal fibroblasts (HPFBs) for the expression and regulation of C-X-C and C-C chemokines. Quiescent HPFBs secreted monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 constitutively. This release could be dose-dependently augmented with the pro-inflammatory cytokines IL-1beta and tumor necrosis factor-alpha. Stimulated IL-8 production reached a plateau within 48 hours while MCP-1 continued to accumulate throughout 96 hours. Induction of IL-8 and MCP-1 synthesis by HPFBs was also triggered by peritoneal macrophage-conditioned medium. This effect was partly related to the presence of IL-1beta as demonstrated by IL-1 receptor antagonist inhibition. Pretreatment of HPFBs with actinomycin D or puromycin dose-dependently reduced cytokine-stimulated IL-8 and MCP-1 secretion, which suggested de novo chemokine synthesis. Indeed, exposure of HPFBs to IL-1beta and tumor necrosis factor-alpha produced a significant up-regulation of IL-8 and MCP-1 mRNA. This effect was associated with the rapid induction of nuclear factor-kappaB binding activity mediated through p65 and p50 subunits, and with a transient increase in the mRNA expression for RelB and inhibitory protein kappaB-alpha proteins. These data indicate that peritoneal fibroblasts are capable of generating large quantities of chemokines under a tight control of nuclear factor-kappaB/Rel transcription factors. Thus, peritoneal fibroblast-derived chemokines may contribute to the intraperitoneal recruitment of leukocytes during peritonitis.

Recombinant soluble p-selectin glycoprotein ligand-1-Ig reduces restenosis through inhibition of platelet-neutrophil adhesion after double angioplasty in swine.

P-selectin mediates leukocyte recruitment to activated platelets and endothelium through its high-affinity receptor P-selectin glycoprotein ligand-1 (PSGL-1). Platelet and leukocyte activation and binding have been reported after coronary angioplasty and were correlated with restenosis. We investigated the effect of a recombinant soluble PSGL-1 (rPSGL-Ig) on the adhesion of platelets and neutrophils and the development of restenosis after double arterial injury. Four weeks after angioplasty of both carotid arteries in pigs, a second angioplasty was performed at the same sites, 15 minutes after a single administration of vehicle or rPSGL-1 (1 mg/kg IV). Animals were euthanized 1 hour, 4 hours, 1 week, or 4 weeks later. Adhesion of autologous (51)Cr-platelets and (111)In-neutrophils was quantified and histological/morphometric analyses were performed. Although rPSGL-Ig did not affect adherence of these cells 1 hour after injury, it significantly reduced the adhesion of platelets (50% at 4 hours and 85% at 1 week) and neutrophils (50% at 4 hours and 78% at 1 week) to deeply injured arteries. At 4 weeks, the residual lumen was 63% larger in rPSGL-Ig-treated arteries as compared with control arteries (6.1+/-0.6 versus 3.8+/-0.1 mm(2); P:<0.002). The neointimal area was slightly reduced (0.5 in rPSGL-Ig versus 0.7 mm(2) in control). The ratio of the external elastic lamina of injured to uninjured reference segments was >1 in treated arteries and <1 in control arteries. P-selectin antagonism with rPSGL-Ig inhibits early platelet/leukocyte adhesion on injured arteries and reduces restenosis through a positive impact on vascular remodeling. Hence, rPSGL-Ig may have potential in the prevention of restenosis.

L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo.

L-selectin has been shown to be important in mediating leukocyte recruitment during inflammatory responses. Although there are numerous in vitro studies demonstrating that engagement of L-selectin leads to the activation of several signaling pathways potentially contributing to subsequent adhesion, emigration, or even migration through the interstitium, whether this actually induces cellular events in vivo is completely unknown. Therefore, we used intravital microscopy to visualize the role of L-selectin in downstream leukocyte adhesion, emigration, and interstitial migration events in wild-type and L-selectin-deficient (L-selectin(-/-)) mice. The cremaster muscle was superfused with the chemotactic inflammatory mediators platelet-activating factor or KC. Leukocyte rolling, adhesion, and emigration in postcapillary venules were examined, and the migration of emigrated leukocytes was recorded continuously using time-lapse videomicroscopy. Platelet-activating factor increased leukocyte adhesion to a similar level in both wild-type and L-selectin(-/-) mice. In contrast, both the number of emigrated leukocytes and the distance of extravascular migration were significantly reduced in L-selectin(-/-) mice. A similar pattern was observed in response to the superfusion of KC. Because superfusion of these mediators induced chemokinesis, we developed a new in vivo chemotaxis assay using slow release of KC from an agarose gel positioned 350 microm from a postcapillary venule. These experiments showed that L-selectin(-/-) leukocytes were also severely impaired in their ability to respond to a directional cue. These findings indicate that L-selectin is important in enabling leukocytes to respond effectively to chemotactic stimuli in inflamed tissues.

A role for platelets and endothelial selectins in tumor necrosis factor-alpha-induced leukocyte recruitment in the brain microvasculature.

The mechanisms mediating leukocyte recruitment into the cerebral nervous system during inflammation are still poorly understood. The objective of this study was to investigate the leukocyte recruitment in the brain microcirculation by intravital microscopy. Superfusion of the brain with artificial cerebrospinal fluid did not induce leukocyte rolling or adhesion. However, intraperitoneal tumor necrosis factor-alpha (TNF-alpha) caused marked leukocyte rolling and adhesion in the brain microcirculation. Histology revealed that the recruitment was primarily of neutrophils. Both E- and P-selectin were required for TNF-alpha-induced leukocyte recruitment, as rolling was reduced after treatment with either anti-E- or anti-P-selectin antibody and eliminated in E- or P-selectin-deficient mice. A significant increase in brain P- and E-selectin expression was seen after TNF-alpha treatment, but both were an order of magnitude less than in any other tissue. We observed significant platelet paving of TNF-alpha-stimulated endothelium and found that anti-platelet antibody reduced leukocyte rolling and adhesion, as did acetylsalicylic acid (aspirin). However, depletion of platelets did not reduce cerebral P-selectin expression. Moreover, chimeric mice lacking P-selectin on endothelium but not platelets had significantly decreased P-selectin expression and reduced leukocyte recruitment in the brain. This suggests a role for endothelial P-selectin in cerebral leukocyte recruitment. In conclusion, TNF-alpha-induced neutrophil recruitment into the brain requires both endothelial E-selectin and P-selectin as well as platelets, but platelet P-selectin was not a major contributor to this process.

The role of macrophage inflammatory protein-1 alpha/CCL3 in regulation of T cell-mediated immunity to Cryptococcus neoformans infection.

Macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3) is a CC chemokine required for optimal recruitment of leukocytes in response to cryptococcal Ags. MIP-1alpha is expressed in the lungs by day 6 post Cryptococcus neoformans infection and could play a role in the development of cell-mediated immunity. To address this possibility, wild-type (MIP-1alpha(+/+)) mice and MIP-1alpha knockout (MIP-1alpha(-/-)) mice were infected intratracheally with a highly virulent strain of C. neoformans (145A). MIP-1alpha message was detected in the lungs on days 3, 7, and 14 in MIP-1alpha(+/+) mice, but it was undetectable in MIP-1alpha(-/-) mice. On day 16, MIP-1alpha(-/-) mice had a 7-fold increase in C. neoformans burden in the lungs, but no decrease in pulmonary leukocyte recruitment. MIP-1alpha(+/+) and MIP-1alpha(-/-) mice had similar numbers of recruited lymphocytes and monocytes/macrophages. Notably, MIP-1alpha(-/-) mice had a significantly greater number of eosinophils. MIP-1alpha(-/-) mice had extremely high levels of serum IgE. This switch of immune response to a T(2) phenotype was associated with enhanced IL-4 and IL-13 expression in the lungs of MIP-1alpha(-/-) mice compared with MIP-1alpha (+/+) mice. Progression of pulmonary cryptococcosis in the presence of nonprotective T(2) immunity resulted in profound lung damage in MIP-1alpha(-/-) mice (eosinophilic crystal deposition, destruction of lung parenchyma, and pulmonary hemorrhage). Twelve-week survival was dramatically decreased in MIP-1alpha(-/-) mice. These studies, together with our previous studies, demonstrate that MIP-1alpha plays a role in both the afferent (T(1)/T(2) development) and efferent (T(1)-mediated leukocyte recruitment) phases of cell-mediated immunity to C. neoformans.

VLA-4 (α4β1) engagement defines a novel activation pathway for β2 integrin–dependent leukocyte adhesion involving the urokinase receptor

During acute inflammatory processes, β2 and β1 integrins sequentially mediate leukocyte recruitment into extravascular tissues. We studied the influence of VLA-4 (very late antigen-4) (4β1) engagement on β2 integrin activation-dependent cell-to-cell adhesion. Ligation of VLA-4 by the soluble chimera fusion product vascular cell adhesion molecule-1 (VCAM-1)–Fc or by 2 anti-CD29 (β1 chain) monoclonal antibodies (mAb) rapidly induced adhesion of myelomonocytic cells (HL60, U937) to human umbilical vein endothelial cells (HUVECs). Cell adhesion was mediated via β2 integrin (LFA-1 and Mac-1) activation: induced adhesion to HUVECs was inhibited by blocking mAbs anti-CD18 (70%-90%), anti-CD11a (50%-60%), or anti-CD11b (60%-70%). Adhesion to immobilized ligands of β2 integrins (intercellular adhesion molecule-1 [ICAM-1], fibrinogen, keyhole limpet hemocyanin) as well as to ICAM-1–transfected Chinese hamster ovary cells, but not to ligands of β1 integrins (VCAM-1, fibronectin, laminin, and collagen), was augmented. VCAM-1–Fc binding provoked the expression of the activation-dependent epitope CBRM1/5 of Mac-1 on leukocytes. Clustering of VLA-4 through dimeric VCAM-1–Fc was required for β2 integrin activation and induction of cell adhesion, whereas monovalent VCAM-1 or Fab fragments of anti-β1 integrin mAb were ineffective. Activation of β2 integrins by 4β1 integrin ligation (VCAM-1–Fc or anti-β1 mAb) required the presence of urokinase receptor (uPAR) on leukocytic cells, because the removal of uPAR from the cell surface by phosphatidylinositol-specific phospholipase C reduced cell adhesion to less than 40%. Adhesion was reconstituted when soluble recombinant uPAR was allowed to reassociate with the cells. Finally, VLA-4 engagement by VCAM-1–Fc or anti-β1 integrin mAb induced uPAR-dependent adhesion to immobilized vitronectin as well. These results elucidate a novel activation pathway of β2 integrin–dependent cell-to-cell adhesion that requires 4β1 integrin ligation for initiation and uPAR as activation transducer.

VLA-4 (α4β1) engagement defines a novel activation pathway for β2 integrin–dependent leukocyte adhesion involving the urokinase receptor

AbstractDuring acute inflammatory processes, β2 and β1 integrins sequentially mediate leukocyte recruitment into extravascular tissues. We studied the influence of VLA-4 (very late antigen-4) (α4β1) engagement on β2 integrin activation-dependent cell-to-cell adhesion. Ligation of VLA-4 by the soluble chimera fusion product vascular cell adhesion molecule-1 (VCAM-1)–Fc or by 2 anti-CD29 (β1 chain) monoclonal antibodies (mAb) rapidly induced adhesion of myelomonocytic cells (HL60, U937) to human umbilical vein endothelial cells (HUVECs). Cell adhesion was mediated via β2 integrin (LFA-1 and Mac-1) activation: induced adhesion to HUVECs was inhibited by blocking mAbs anti-CD18 (70%-90%), anti-CD11a (50%-60%), or anti-CD11b (60%-70%). Adhesion to immobilized ligands of β2 integrins (intercellular adhesion molecule-1 [ICAM-1], fibrinogen, keyhole limpet hemocyanin) as well as to ICAM-1–transfected Chinese hamster ovary cells, but not to ligands of β1 integrins (VCAM-1, fibronectin, laminin, and collagen), was augmented. VCAM-1–Fc binding provoked the expression of the activation-dependent epitope CBRM1/5 of Mac-1 on leukocytes. Clustering of VLA-4 through dimeric VCAM-1–Fc was required for β2 integrin activation and induction of cell adhesion, whereas monovalent VCAM-1 or Fab fragments of anti-β1 integrin mAb were ineffective. Activation of β2 integrins by α4β1 integrin ligation (VCAM-1–Fc or anti-β1 mAb) required the presence of urokinase receptor (uPAR) on leukocytic cells, because the removal of uPAR from the cell surface by phosphatidylinositol-specific phospholipase C reduced cell adhesion to less than 40%. Adhesion was reconstituted when soluble recombinant uPAR was allowed to reassociate with the cells. Finally, VLA-4 engagement by VCAM-1–Fc or anti-β1 integrin mAb induced uPAR-dependent adhesion to immobilized vitronectin as well. These results elucidate a novel activation pathway of β2 integrin–dependent cell-to-cell adhesion that requires α4β1 integrin ligation for initiation and uPAR as activation transducer.

Prominent role of P-selectin in the development of advanced atherosclerosis in ApoE-deficient mice.

Adhesive interactions between leukocytes and endothelial cells are characteristic of the development of atherosclerotic lesions, but the receptors involved remain to be defined. P-selectin is an adhesion receptor expressed on activated endothelial cells or platelets and was shown to be involved in fatty streak formation in LDL receptor-deficient mice on an atherogenic diet. The main purpose of this study is to examine the role of P-selectin in the spontaneous development of advanced atherosclerosis in apoE-deficient mice. METHODDS AND RESULTS: We intercrossed P-selectin-deficient mice with mice lacking apoE and compared lesion development in apoE-deficient mice with P-selectin (apoE(-/-) P(+/+)) and without P-selectin (apoE(-/-) P(-/-)) that were fed normal mouse chow. At 4 months of age, apoE(-/-) P(-/-) mice had 3. 5-fold smaller aortic sinus lesions than apoE(-/-) P(+/+) mice. These were limited to fatty streaks in the apoE(-/-) P(-/-) mice, whereas 70% of apoE(-/-) P(+/+) lesions contained smooth muscle cells. Significantly more of the aortic sinus circumference was covered by lesions in the apoE(-/-) P(+/+) animals. The P-selectin genotype affected macrophage recruitment, because twice as many mononuclear cells were present in the P-selectin-positive lesions. At 15 months, the lesions progressed to the fibrous plaque stage in both genotypes and spread throughout the aorta, but this process was delayed in apoE(-/-) P(-/-) mice. In the aortic sinus, the lesions of the apoE(-/-) P(-/-) mice were 2.6-fold smaller and less calcified. P-selectin appears to be a key adhesion receptor mediating leukocyte recruitment into lesions and promoting advanced atherosclerosis in apoE-deficient mice.